This invention relates to recombinant DNA technology. In particular the invention pertains to the isolation of novel genes and proteins that encode inositolphosphoryl ceramide synthase (IPC) synthase or subunit thereof from a variety of fungi and the use of said proteins in screens for inhibitors of IPC Synthase.
The incidence of life-threatening fungal infections is increasing at an alarming rate. With the exception of Staphylococci infections, the fungus C. albicans represents the fastest growing area of concern in hospital acquired bloodstream infections. About 90% of nosocomial fungal infections are caused by species of Candida with the remaining 10% being attributable to infections by Aspergillus, Cryptococcus, and Pneumocystis. While effective antifungal compounds have been developed for Candida there is growing concern that the rise in the incidence of fungal infections may portend greater resistance and virulence in the future. Moreover, anti-Candida compounds frequently do not possess clinically significant activity against other fungal species.
Inositolphosphoryl ceramides are sphingolipids found in a number of fungi including but not limited to S. cerevisiae, S. pombe, C. albicans, A. fumigatus, A. nidulans and H. capsulatum. A step of sphingolipid biosynthesis unique to fungi and plants is catalyzed by the enzyme IPC synthase. The IPC synthase step, covalently links inositol phosphate and ceramide, and is essential for viability in S. cerevisiae. Although some elements of sphingolipid biosynthesis in fungi are shared with mammalian systems, the pathways diverge at the step after formation of ceramide. Thus, the formation of inositolphosphoryl ceramide is unique to fungi and plants, making IPC synthase a good molecular target for antifungal chemotherapy.
While compounds that target IPC synthase bode well for the future of anti-fungal therapy, presently there are no clinically useful compounds that act at this step. Thus, there is a need for new compounds that inhibit IPC synthase.
The present invention relates to fungal IPC synthase and to screens for inhibitors thereof.
In one embodiment the invention relates to fungal genes that encode IPC synthase, or subunit thereof.
In another embodiment, the invention relates to fungal IPC synthase genes identified herein as SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:19, and SEQ ID NO:20.
In another embodiment, the invention relates to nucleic acids that are at least 70% homologous, and/or, will hybridize under high stringency conditions to a sequence identified herein as SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:19, and SEQ ID NO:20.
In another embodiment the present invention pertains to the proteins produced by IPC synthase genes.
In yet another embodiment, the invention relates to proteins designated herein as SEQ ID NO 2, SEQ ID NO 5, SEQ ID NO 8, SEQ ID NO 11, and SEQ ID NO:21.
In still another embodiment the invention relates to the use of purified fungal IPC synthase or subunit thereof in high throughput screens for inhibitors of fungal IPC synthase, said IPC synthase being designated herein as SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, and SEQ ID NO:21.
In another embodiment the invention relates to the use of recombinant host cells that carry a vector that expresses a fungal IPC synthase in high throughput screens for inhibitors of fungal IPC synthase.
In another embodiment the invention relates to the use of the IPC synthase genes disclosed herein, or fragments thereof, as hybridization probes or PCR primers to identify and isolate homologous genes that are related in sequence and/or function.
The terms xe2x80x9ccleavagexe2x80x9d or xe2x80x9crestrictionxe2x80x9d of DNA refers to the catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA (viz. sequence-specific endonucleases). The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors, and other requirements are used in the manner well known to one of ordinary skill in the art. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer or can readily be found in the literature.
The term xe2x80x9cfusion proteinxe2x80x9d denotes a hybrid protein molecule not found in nature comprising a translational fusion or enzymatic fusion in which two or more different proteins or fragments thereof are covalently linked on a single polypeptide chain.
The term xe2x80x9cplasmidxe2x80x9d refers to an extrachromosomal genetic element. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accordance with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
xe2x80x9cRecombinant DNA cloning vectorxe2x80x9d as used herein refers to any autonomously replicating agent, including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DNA segments can or have been added.
The term xe2x80x9crecombinant DNA expression vectorxe2x80x9d as used herein refers to any recombinant DNA cloning vector, for example a plasmid or phage, in which a promoter and other regulatory elements are present to enable transcription of the inserted DNA.
The term xe2x80x9cvectorxe2x80x9d as used herein refers to a nucleic acid compound used for introducing exogenous DNA into host cells. A vector comprises a nucleotide sequence which may encode one or more protein molecules. Plasmids, cosmids, viruses, and bacteriophages, in the natural state or which have undergone recombinant engineering, are examples of commonly used vectors.
The terms xe2x80x9ccomplementaryxe2x80x9d or xe2x80x9ccomplementarityxe2x80x9d as used herein refers to the capacity of purine and pyrimidine nucleotides to associate through hydrogen bonding in double stranded nucleic acid molecules. The following base pairs are complementary: guanine and cytosine; adenine and thymine; and adenine and uracil.
xe2x80x9cIsolated nucleic acid compoundxe2x80x9d refers to any RNA or DNA sequence, however constructed or synthesized, which is locationally distinct from its natural location.
A xe2x80x9cprimerxe2x80x9d is a nucleic acid fragment which functions as an initiating substrate for enzymatic or synthetic elongation of, for example, a nucleic acid molecule.
The term xe2x80x9cpromoterxe2x80x9d refers to a DNA sequence which directs transcription of DNA to RNA.
A xe2x80x9cprobexe2x80x9d as used herein is a nucleic acid compound that hybridizes with another nucleic acid compound, and is useful for blot hybridizations, for example. A probe is at least 15 base pairs in length, its sequence being at least 90% identical with the nucleic acid molecules disclosed herein, or fragments thereof, or the complements thereof. A probe may or may not be labeled with a detectable moiety. As used herein, a probe is useful for hybridization analysis to identify sequences homologous to those disclosed herein.
The term xe2x80x9chybridizationxe2x80x9d as used herein refers to a process in which a single-stranded nucleic acid molecule joins with a complementary strand through nucleotide base pairing. xe2x80x9cSelective hybridizationxe2x80x9d refers to hybridization under conditions of high stringency. The degree of hybridization depends upon, for example, the degree of complementarity, the stringency of hybridization, and the length of hybridizing strands.
The term xe2x80x9cstringencyxe2x80x9d refers to hybridization conditions. High stringency conditions disfavor non-homologous basepairing. Low stringency conditions have the opposite effect. Stringency may be altered, for example, by temperature and salt concentration.
xe2x80x9cLow stringencyxe2x80x9d conditions comprise, for example, a temperature of about 37xc2x0 C. or less, a formamide concentration of less than about 50%, and a moderate to low salt (SSC) concentration; or, alternatively, a temperature of about 50xc2x0 C. or less, and a moderate to high salt (SSPE) concentration.
xe2x80x9cHigh stringencyxe2x80x9d conditions comprise a temperature of about 42xc2x0 C. or less, a formamide concentration of less than about 20%, and a low salt (SSC) concentration; or, alternatively, a temperature of about 65xc2x0 C., or less, and a low salt (SSPE) concentration.
xe2x80x9cSSCxe2x80x9d comprises a hybridization and wash solution. 20X SSC contains 3M sodium chloride, 0.3M sodium citrate, pH 7.0.
xe2x80x9cSSPExe2x80x9d comprises a hybridization and wash solution. 1X SSPE contains 180 mM NaCl, 9mM Na2HPO4,0.9 mM NaH2PO4 and 1 mM EDTA, pH 7.4.